Angiogenesis (also referred to herein as neovascularization) is the process whereby new blood vessels are formed. Angiogenesis occurs normally during embryogenesis and development, and occurs in fully developed organisms during wound healing and placental development. In addition, angiogenesis occurs in various pathological conditions, including in ocular diseases such as diabetic retinopathy and macular degeneration due to neovascularization, in conditions associated with tissue inflammation such as rheumatoid arthritis and inflammatory bowel disease, and in cancer, where blood vessel formation in the growing tumor provides oxygen and nutrients to the tumor cells, as well as providing a route via which tumor cells metastasize throughout the body. Since millions of people around the world are afflicted by these diseases, a considerable effort has been made to understand the mechanisms involved in angiogenesis in the hope that such an understanding will allow the development of methods for detecting and inhibiting such undesirable angiogenesis.
Angiogenesis occurs in response to stimulation by one or more known growth factors, and also may involve other as yet unidentified factors. Endothelial cells, which are the cells that line mature blood vessels, normally do not proliferate. However, in response to an appropriate stimulus, the endothelial cells become activated and begin to proliferate and migrate into unvascularized tissue forming new blood vessels. In some cases, precursor cells can be activated to differentiate into endothelial cells which form new blood vessels.
Blood vessels are surrounded by an extracellular matrix. In addition to stimulation by growth factors, neovascularization depends on interaction of the endothelial cells with the extracellular matrix, as well as with each other. The activation of endothelial cells by growth factors and the migration into and interaction with the extracellular matrix and with each other is dependent on cell surface receptors expressed by the endothelial cells. These cell surface receptors, which include growth factor receptors and integrins, interact specifically with particular molecules.
In pathological conditions such as age-related macular degeneration and diabetic retinopathy, decreasing availability of oxygen to the retina results in a hypoxic condition that stimulates the secretion of angiogenic growth factors such as vascular endothelial growth factors (VEGF), which induce abnormal migration and proliferation of endothelial cells into tissues of the eye. Such neovascularization in ocular tissues can induce corneal scarring, retinal detachment and fluid accumulation in the choroid, each of which can adversely affect vision and lead to blindness.
Angiogenesis also is associated with the progression and exacerbation of inflammatory diseases, including psoriasis, rheumatoid arthritis, osteoarthritis, and inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. In inflammatory arthritic disease, for example, influx of lymphocytes into the region surrounding the joints stimulates angiogenesis in the synovial lining. The increased vasculature provides a means for greater influx of leukocytes, which facilitate the destruction of cartilage and bone in the joint. Neovascularization that occurs in inflammatory bowel disease results in similar effects in the bowel.
The growth of capillaries into atherosclerotic plaques in the coronary arteries represents another pathological condition associated with growth factor induced angiogenesis. Excessive blood flow into neovascularized plaques can result in rupture and hemorrhage of the blood-filled plaques, releasing blood clots that can result in coronary thrombosis.
The involvement of angiogenesis in such diverse diseases as cancer, ocular disease and inflammatory diseases has led to an effort to identify methods for specifically inhibiting angiogenesis as a means to treat these diseases. For cancer patients, such methods of treatment can provide a substantial advantage over currently used methods such as chemotherapy, which kill or impair not only the target tumor cells, but also normal cells in the patient, particularly proliferating normal cells such as blood cells, epithelial cells, and cells lining the intestinal lumen. Such non-specific killing by chemotherapeutic agents results in side effects that are, at best, unpleasant, and can often result in unacceptable patient morbidity, or mortality. In fact, the undesirable side effects associated with cancer therapies often limit the treatment a patient can receive.
For other pathological conditions associated with abnormal angiogenesis such as diabetic retinopathy, there are no effective treatments short of retinal transplants. However, even if retinal transplantation is performed, the new retina would be subject to the same conditions that resulted in the original retinopathy. Thus, there exists a need for novel methods of inhibiting and treating neovascularization in patients suffering from pathological conditions characterized by this condition. The present invention satisfies this need and provides related advantages as well in the treatment of other disease conditions identified herein.
The retina is the part of the eye that is sensitive to light. The macula lutea is the region of the retina that allows us to read and recognize faces. Diseases of the macula, such as age-related macular degeneration (AMD) and diabetic macular edema, account for a major proportion of legal blindness. To combat these diseases, a variety of accepted and experimental medications are employed via systemic routes or local, invasive surgical procedures.
Diabetic retinopathy (DR), a leading cause of blindness in type 1 and type 2 diabetics, is a complication of diabetes which produces damage to the blood vessels inside the retina. Diabetic retinopathy can have four stages: (1) mild nonproliferative retinopathy, wherein microaneurysms in the retina's blood vessels occur; (2) moderate nonproliferative retinopathy, wherein some blood vessels feeding the retina become blocked; (3) severe nonproliferative retinopathy, wherein many blood vessels to the retina are blocked, depriving several areas of the retina with their blood supply; and (4) proliferative retinopathy, wherein new, abnormal, thin-walled and fragile-walled blood vessels grow to supply blood to the retina, but which new blood vessels may leak blood to produce severe vision loss and blindness. Hemorrhages can occur more than once, often during sleep. Fluid can also leak into the center of the macula at any stage of diabetic retinopathy and cause macular edema and blurred vision. About 40 to 45 percent of Americans diagnosed with diabetes have some stage of diabetic retinopathy, and about half of the people with proliferative retinopathy also have macular edema.
Macular degeneration is a degeneration of the macular region of the retina in the eye. Degeneration of the macula causes a decrease in acute vision and can lead to eventual loss of acute vision. The wet form of macular degeneration is related to abnormal growth of blood vessels in the retina that can leak blood and can cause damage to photoreceptor cells. Age-related macular degeneration is a collection of clinically recognizable ocular findings that can lead to blindness. Macular degeneration is a group of diseases. There are two basic types of macular degeneration, including “wet” and “dry”. In wet macular degeneration, there is an abnormal growth of new blood vessels (neovascularization). These new blood vessels break and leak fluid, causing damage to the central retina. This form of macular degeneration is often associated with aging. Approximately 85% of macular degeneration cases are dry macular degeneration. Vision loss can result from the accumulation of deposits in the retina called druzen, and from the death of photoreceptor cells in the retina. This process can lead to thinning and drying of the retina.
The findings of AMD include the presence of druzen, retinal pigment epithelial disturbance, including pigment clumping and/or dropout, retinal pigment epithelial detachment, geographic atrophy, subretinal neovascularization and disciform scar. Age-related macular degeneration is a leading cause of presently incurable blindness, particularly in persons over 55 years of age. Approximately one in four persons age 65 or over have signs of age-related maculopathy, and about 7% of persons age 75 or over have advanced macular degeneration with vision loss.
Druzen are opthalmoscopically visible, yellow-white hyaline excrescences or nodules of Bruch's membrane. Bruch's membrane lies beneath the retina and the adjacent retina pigment epithelium layer. Fat accumulates in Bruch's membrane with age and may contribute to the formation of druzen. Druzen can occur in two forms. One form comprises hard, small (less than about 60 micrometers in diameter) objects which do not increase with age and which do not predispose to macular degeneration. Another form comprises soft, large (more than about 63 micrometers in diameter) objects which enlarge and become confluent with age. The soft, large druzen may predispose to macular degeneration, and are commonly seen in eyes of people with advanced macular degeneration in at least their other eye.
Druzen may be metabolic waste products from various layers of the retina such as from the retina, retina pigment epithelium, and choriocapillaris. Druzen may be yellow, white, gray, retractile, and/or pink. Druzen may be small, medium or large in size. Druzen may be regular or irregular, or symmetrical or asymmetrical in shape. A patient who has druzen and who suffers complications in one eye may suffer no complications in the other eye. Complications may comprise one or more conditions selected from the group consisting of retina pigment epithelium atrophy, choroid neovascularization, retina detachment serous, and retina detachment hemorrhagic. Druzen may affect contrast sensitivity, and may reduce the eye's ability to see sufficiently to allow a person to read in dim light or to see sufficient detail to permit a person to drive an automobile safely at night.
A contributing and indicating factor of advanced macular degeneration is neovascularization of the choroid tissue underlying the photoreceptor cells in the macula. Healthy mature ocular vasculature is normally quiescent and exists in a state of homeostasis in which a balance is maintained between positive and negative mediators of angiogenesis in development of new vasculature. Macular degeneration, particularly in its advanced stages, is characterized by the pathological growth of new blood vessels in the choroid underlying the macula. Angiogenic blood vessels in the subretinal choroid can leak vision obscuring fluids, leading to blindness.
The major causes of blindness in the United States are glaucoma, AMD, cataracts, DR, and retinitis pigmentosa (RP) which translates into more than 38 million citizens having some form of an age related eye disease. In developed countries, cataracts are routinely surgically removed and vision is restored with the insertion of an artificial lens. There are no cures for most forms of the other blinding diseases, the severity of which increases with age and dramatically decreases the quality of life for these patients. Even for patients with an inherited blindness, such as RP, vision worsens with age. Nearly 1 out of 3 individuals over the age of 75 will develop some form of AMD and with the aging of the “Baby Boomers” generation, this means a dramatic increase in patient numbers. Nearly 200,000 individuals in the USA develop AMD each year. About 2 million Americans over the age of 40 have significant vision loss due to AMD while an additional 8 million have a high risk of vision loss. DR has some symptoms very similar to wet AMD including neovascular growth in the eye and subfoveal macular edema. About 4 million Americans age 40 and older have DR and >80% of patients who have diabetes for more than ten years will develop DR. Further, because Native American Indians develop diabetes at a much higher rate than the general population, DR is becoming a progressively increasing problem in states such as Oklahoma which have large numbers of Native Americans. The annual economic cost to the USA for adult vision loss is major at about $50 billion per year.
As noted above, AMD is classified roughly into two categories based on the absence or presence of choroidal neovascularizations which grow into the eye. Most patients have the dry form (85%) whereas about 15% have the wet form. The dry form is characterized by the accumulation of debris (druzen) between the retinal pigment epithelia and Bruch's membrane which effectively eliminates the benefits of the choroidal blood supply to the adjacent photoreceptor cells. The dry form can lead to the wet form, but optionally may not, and patients may still retain some retinal function throughout life. In the wet form, the presence of sub-macular neovascular vessels leads to retinal edema, ruptured blood vessels, the death of the cones in the macula and eventual blindness. Current therapeutic treatments for wet AMD involve intravitreal injections of monoclonal antibodies against Vascular Endothelial Growth Factor (VEGF) every 6-8 weeks. There are no treatments which have proven successful for dry AMD. Recently, a subform of AMD was recognized, called Retinal Angiomatous Proliferation (RAP), in which neovascular lesions occurred within the photoreceptor cell layer as a result of neovessels growing from the retinal vasculature through the photoreceptors, the RPE and Bruch's membrane where they joined choroidal neovascular tufts. These patients represent about 15% of the vascular form of AMD. It is an object of the present invention to develop a therapeutic treatment for blinding diseases, such as RAP.
Mammalian cells produce cellular energy in mitochondria by using oxygen to metabolize molecular substrates. The vast majority of the products of this oxidative metabolism are beneficial while about 3% are highly toxic compounds such as singlet oxygen, the hydroxide ion, and hydrogen peroxide. These Reactive Oxygen Species (ROS) can react with and damage almost any type of molecule within the cell including proteins, DNA, RNA and lipids. Another major source of intracellular ROS is NADPH oxidase which activates the STAT3 pathway which upregulates retinal VEGF. The normal antioxidant cellular defenses against ROS include catalytic proteins such as superoxide dismutase, heme oxygenase and thioredoxin as well as small molecules like glutathione, and NADPH. Oxidative stress occurs when the level of ROS exceeds the ability of the cells' antioxidant defenses to scavenge or destroy them. Because of the close proximity of the intra-mitochondrial components to the ROS, it is not surprising that they bear the brunt of damage from ROS and with decreased oxidative phosphorylation they produce less energy but more ROS.
As indicated herein, there are many diseases which result in the programmed cell death of photoreceptor cells and blindness. These include illnesses which are known to be inherited such as retinitis pigmentosa as well as many which have a genetic component but which may be environmentally induced or are of questionable origin such as diabetic retinopathy and AMD. Interestingly, irrespective of the primary cause, all of these diseases are thought to share some common nodes, including oxidative stress caused by a chronic or acute rise in ROS and apoptosis. The retina has the highest rate of oxygen metabolism, is constantly bombarded with photons of light, and is therefore exposed to a higher concentration of ROS than any other tissue of the body. Neurodegeneration within the retina is not unlike neurodegeneration within other areas of the central nervous system. Even in the albino rat model of light-induced degeneration of photoreceptor cells, initiation of apoptosis proceeds through the intracellular production of ROS. Strong evidence that oxidative damage is a primary cause of AMD was recently presented by Hollyfield et al. (“Oxidative damage-induced inflammation initiates age-related macular degeneration’, Nat. Med. 2008; 14:194-198).